Neisseria gonorrhoeae (the gonococcus; [Gc]) causes both localized, uncomplicated infections at mucosal surfaces and more invasive forms of disease that can have severe medical consequences for the reproductive and general health of men and women. In the United States it is estimated that the incidence of gonorrhea is at least 850,000 cases per year while the worldwide incidence in 2012 was reported to be 78 million cases. In addition to the global problems of disease incidence and impact of gonorrhea on human health, Gc strains have developed decreased susceptibility or clinical resistance to front-line antibiotics currently used in dual therapy (e.g., ceftriaxone and azithromycin) in the USA and other developed countries. Our research program is dedicated to understanding the intrinsic systems possessed by Gc that allow it to resist classical antibiotics as well as cationic antimicrobial peptides/proteins (CAMPs) that participate in innate host defense. In the absence of new antibiotics, especially those recognizing unexploited targets, information regarding the development of resistance to existing antibiotics likely to enter clinical practice is critical. For instance, gentamicin has been used extensively in Africa to treat gonorrhea but resistance data from this part of the world is scant. This is unfortunate because gentamicin has been suggested to be an alternative antibiotic for treatment of gonorrhea in developed countries. We have evidence that loss of a sensory two component system (TCS) termed MisR/MisS results in Gc hyper-susceptibility to both gentamicin (as well as other aminoglycosides) and CAMPs, but not other antibiotics. A recent report documented that loss of the MisR/MisS TCS conferred a survival defect on Gc as assessed in a female mouse model of lower genital tract infection. We hypothesize that such attenuation of Gc was due, in part, to a reduced capacity of MisR-negative Gc to regulate genes important for resistance to host-derived CAMPs. The selective sensitivity of MisR-negative Gc to aminoglycosides and CAMPs is consistent with the self-promoted uptake model invoked for how these antimicrobials enter Pseudomonas aeruginosa and traverse the cell envelope. We will now build on the progress made in our initial studies. In Specific Aim 1 we will use genetic screening techniques, an experimental female mouse infection model and analysis of clinical isolates expressing decreased gentamicin susceptibility to further define the repertoire of Gc genes that contribute to intrinsic gentamicin resistance. We hypothesize that the identified genes will encode proteins that participate in cell envelope structure, membrane integrity and membrane homeostasis that work in unison to impact levels of Gc resistance to aminoglycosides and CAMPs. In Specific Aim 2 we will ascertain the responsiveness of Gc to sub-lethal levels of gentamicin and determine the responsible genes. In Specific Aim 3 we will define the physiological consequences of mutations in the identified genes that influence Gc susceptibility to gentamicin. The results from this work will provide basic knowledge as to how human pathogens like Gc control expression of genes involved in intrinsic resistance to antimicrobials.